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. 2020 Nov 25;10(12):1598. doi: 10.3390/biom10121598

Figure 1.

Figure 1

(A) Laf1 co-localizes with Lam2 at ER-PM CS contact sites. A strain (yEPS23) co-expressing Laf1-mKate, GFP-Lam2, and Pil1-GFP, each from its respective endogenous chromosomal locus, was grown to mid-exponential phase and viewed directly under an epifluorescence microscope, as described in the Materials and Methods Section. (B) Localization of Lam2 and Laf1 to ER-PM CSs is reduced, but not abolished, in the tether∆ strain. A wild-type strain (YFR712) and an otherwise isogenic tether∆ strain (YFR704) expressing Lam2-mNG (left panels) and the wild-type strain (YFR729) and the otherwise isogenic tether∆ strain (YFR705) expressing Laf1-mNG (right panels) were grown to mid-exponential phase in YPD and viewed directly under an epifluorescence microscope, as described in the Materials and Methods Section. (C) Localization of Laf1 to ER-PM CSs requires Lam2 and Lam4. Upper panels, Strains expressing Laf1-mNG in a wild-type (YFR713), lam2∆ (YFR714), lam4∆ (YFR715), or lam2∆ lam4∆ (YFR720) background, were grown to mid-exponential phase and viewed directly under an epifluorescence microscope. Lower panels, samples of equivalent amounts (as judged by the Pgk1 loading control) of extracts of the same cells shown in the upper panel were resolved by SDS-PAGE and analyzed by immunoblotting, as described in the Materials and Methods Section. (D) Localization of Dgr2 to ER-PM CSs requires Lam2 and Lam4. Upper panels, Strains expressing Dgr2-mKate in a wild-type (YFR657-B), lam2∆ (YFR658-B), lam4∆ (YFR659-B), or lam2∆ lam4∆ (YFR650-B) background, were grown to mid-exponential phase and viewed directly under an epifluorescence microscope. Lower panels, samples of equivalent amounts (as judged by the Pgk1 loading control) of extracts of the same cells shown in the upper panel were resolved by SDS-PAGE and analyzed by immunoblotting, as described in the Materials and Methods Section. (E) Lam2 localizes to ER-PM CSs in the absence of Laf1 and Dgr2. A WT strain (YFR512-A) and an otherwise isogenic laf1∆ dgr2∆ derivative (YFR613-A), each expressing GFP-Lam2, were grown to mid-exponential phase and viewed using an Elyra PS.1 structured illumination (SIM) fluorescence microscope.